This invention relates to protective Helicobacter antigens, especially H. pylori antigens, and to the use of these antigens for the treatment and prevention of gastroduodenal disease associated with H. pylori infection in humans.
Helicobacter pylori is a bacterium that infects the stomach lining (or gastric mucosa) of perhaps half the world""s population. Spiral organisms were first microscopically observed in human gastric mucosa in 1906. However, H. pylori was not successfully cultured until 1982. Infection with the organism is usually chronic, and results in continuing inflammation of the gastric mucosa. The infection is often asymptomatic. However, in association with other cofactors, a proportion of infected people go on to develop sequelae including peptic ulceration of the stomach or duodenum, gastric adenocarcinomas and gastric lymphomas. Peptic ulcer treatment studies have shown that cure of H. pylori infection is associated with a dramatic reduction in the relapse rate of this usually chronic disease. Long term infection with H. pylori leads to the development of chronic atrophic gastritis, which has long been recognised as a precursor lesion in the development of gastric cancer. Thus a number of studies have now linked preceding H. pylori infection with an increased risk of developing gastric cancer. Therefore eradication of current infection and prevention of new infection with this organism has the potential to significantly reduce the incidence of diseases that result in considerable morbidity and mortalityl1,2.
Infection with H. pylori is difficult to treat. Current experimental therapies for treating the infection have problems with efficacy and significant levels of adverse effects. There are no prophylactic measures available. A solution to both the prevention and treatment of H. pylori infection would be the development of an immunogenic preparation that, as an immunotherapeutic, treated established infections, and as a vaccine, prevented the establishment of new or recurrent infections. Such a preparation would need to induce effective immune responses to protective antigens, while avoiding inducing responses to self antigens or other potentially harmful immune responses. This may be achieved by identifying the specific protective component or components and formulating immunotherapeutic or vaccine preparations including these component(s).
The identification of such protective components of an organism, is often accomplished through the use of an animal model of the infection. Initially, H. pylori did not naturally infect laboratory animals. However, an animal model of human H. pylori infection has been developed using a closely related organism, H. felis, and specific pathogen free (SPF) mice3. These organisms are able to colonise the gastric mucosa of SPF mice, where they establish a chronic infection with many of the features of H. pylori infection in humans. H. felis infection in the mice induces a chronic gastritis and a raised immune response. As in the human case, this response is not effective in curing the infection.
This model has been used to demonstrate that oral treatment of H. felis infected mice with a preparation containing disrupted H. pylori cells and cholera toxin as a mucosal adjuvant, can cure a significant portion of infected mice4. This effect is likely to be mediated through an immune response to a cross-reactive antigen possessed by each of the closely related species.
In working by the inventors leading to the present invention, these cross-reactive antigens were recognised by performing a Western blot using H. pylori disrupted cells as the antigen, and probing the blot with serum from mice immunised with H. felis and cholera toxin adjuvant. Sections of membrane containing proteins recognised as cross-reactive were removed from the membrane, the proteins bound to them were eluted, and their N-terminal amino acid sequence determined by microsequencing.
The N-terminal amino acid sequence of one of the two proteins that successfully yielded sequence data closely matched the previously published sequence of the microbial enzyme, urease5. This enzyme has already been shown to be a protective antigen when used in a vaccine to prevent infection.
The N-terminal amino acid sequence of the other protein closely matched the previously published N-terminal sequence of the microbial enzyme, catalase6. This enzyme has not previously been shown to be a protective antigen of H. pylori. 
International Patent Application No. PCT/FR95/00383 (Publication No. WO 95/27506) in the name Pasteur Merieux Serums et Vaccins, published Oct. 19, 1995, discloses an H. pylori immunising composition, based on proposed use of H. pylori catalase in substantially purified form as an immunising substance useful for prophylactic or therapeutic purposes. It is suggested that the catalase could be obtained either by extraction from H. pylori (using the purification method of Hazell et al.10) or by recombinant means. The disclosure contains no supporting data showing efficacy of H. pylori catalase in use as an immunising substance, nor is there any supporting disclosure or teaching of the preparation of H. pylori catalase by recombinant means or of the efficacy of recombinant catalase in use as an immunising substance.
Recently, an H. pylori (Sydney strain)/mouse model of human H. pylori infection has been developed and used by the present inventors to confirm that catalase, in particular recombinant catalase, has utility as a protective antigen.
In one aspect, the present invention provides an antigenic preparation for use in the treatment or prevention of Helicobacter infection, which comprises an at least partially purified preparation of the catalase of Helicobacter bacteria.
The term xe2x80x9cat least partially purifiedxe2x80x9d as used herein denotes a preparation in which the catalase content is greater, preferably at least 30% and more preferably at least 50% greater, than the catalase content of a whole cell sonicate of Helicobacter bacteria. Preferably, the preparation is one in which the catalase is xe2x80x9csubstantially purexe2x80x9d, that is one in which the catalase content is at least 80%, more preferably at least 90%, of the total Helicobacter antigens in the preparation.
Accordingly, in a particularly preferred embodiment, the present invention provides an antigenic preparation for use in treatment or prevention of Helicobacter infection, which comprises substantially pure catalase of Helicobacter bacteria. Such a preparation may be prepared as a recombinant catalase by techniques described hereinafter.
In another aspect, the present invention provides an isolated Helicobacter antigen for use in the treatment or prevention of Helicobacter infection in a mammalian host, which comprises the catalase of Helicobacter bacteria, or an immunogenic fragment thereof.
The term xe2x80x9cisolatedxe2x80x9d as used herein denotes that the antigen has undergone at least one purification or isolation step, and preferably is in a form suitable for use in a vaccine composition.
It is to be understood that the present invention extends not only to an antigenic preparation or isolated antigen comprising the catalase of Helicobacter bacteria, but also to antigenic preparations comprising immunogenic fragments of this catalase, that is catalase fragments which are capable of eliciting a specific protective immune response in a mammalian host. Such immunogenic fragments may also be recognised by Helicobacter-specific antibodies, particularly monoclonal antibodies which have a protective or therapeutic effect in relation to Helicobacter infection or polyclonal antibodies contained in immune sera from mammalian hosts which have been vaccinated against Helicobacter infection.
In another aspect, the present invention provides a vaccine composition for use in the treatment or prevention of Helicobacter infection in a mammalian host, which comprises an immunologically effective amount of an antigenic preparation or isolated antigen as broadly described above, optionally in association with an adjuvant, together with one or more pharmaceutically acceptable carriers and/or diluents.
In yet another aspect, the present invention provides a method for the treatment or prevention of Helicobacter infection in a mammalian host, which comprises administration to said host of an immunologically effective amount of an antigenic preparation or isolated antigen as broadly described above, optionally in association with an adjuvant.
In a related aspect, this invention provides the use of a vaccine composition comprising an immunologically effective amount of an antigenic preparation or isolated antigen as broadly described above, optionally in association with an adjuvant, for the treatment or prevention of Helicobacter infection in a mammalian host.
In yet another aspect, the invention provides the use of an antigenic preparation or isolated antigen as broadly described above, optionally in association with an adjuvant, in the manufacture of a vaccine composition for the treatment or prevention of Helicobacter infection in a mammalian host.
Preferably, but not essentially, the antigenic preparation or isolated antigen of this invention is orally administered to the host, and is administered in association with a mucosal adjuvant. However, the invention also extends to parenteral administration of this antigenic preparation or isolated antigen.
By use of the term xe2x80x9cimmunologically effective amountxe2x80x9d herein in the context of treatment of Helicobacter infection, it is meant that the administration of that amount to an individual infected host, either in a single dose or as part of a series, that is effective for treatment of Helicobacter infection. By the use of the term xe2x80x9cimmunologically effective amountxe2x80x9d herein in the context of prevention of Helicobacter infection, it is meant that the administration of that amount to an individual host, either in a single dose or as part of a series, that is effective to delay, inhibit or prevent Helicobacter infection. The effective amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the capacity of the individual""s immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
Preferably, the catalase antigen above comprises an amino acid sequence substantially corresponding to the deduced sequence of the catalase gene from isolate RU1 or isolate 921023 hereinafter (SEQ ID NO.2 or 4), or allelic or other variants thereof. Suitable variants may have at least 50-60%, more preferably at least 70-80%, and most preferably at least 90%, similarity to one of the amino acid sequences referred to above, or to a region or part thereof, provided the variant is capable of eliciting a specific protective immune response in a mammalian host.
As described above, the present invention extends not only to the particular catalase antigen of Helicobacter bacteria as described above, but also to immunogenic fragments of the particular antigen, that is fragments of the antigen which are capable of eliciting a specific protective immune response in a mammalian host. Suitably, the immunogenic fragment will comprise at least five, and more preferably at least ten, contiguous amino acid residues of the particular antigen.
Such immunogenic fragments may also be recognised by Helicobacter-specific antibodies, particularly antibodies which have a protective or therapeutic effect in relation to Helicobacter infection.
The present invention also extends to an antibody, which may be either a monoclonal or polyclonal antibody, specific for an antigenic preparation or an isolated Helicobacter antigen as broadly described above. Such antibodies may be produced by methods which are well known to persons skilled in this field.
In this aspect, the invention further provides a method for the treatment or prevention of Helicobacter infection in a mammalian host, which comprises passive immunisation of said host by administration of an effective amount of an antibody, particularly a monoclonal antibody, specific for an antigenic preparation or an isolated Helicobacter antigen as broadly described above.
The Helicobacter antigenic preparation or isolated antigen of this invention may be prepared by purification or isolation from natural sources, such as a whole cell sonicate of Helicobacter bacteria. Alternatively, however the antigenic preparation or isolated antigen may be prepared by synthetic, preferably recombinant, techniques. When prepared by recombinant techniques, the antigen may have an amino acid sequence substantially identical to the naturally occurring sequence or may contain one or more amino acid substitutions, deletions and/or additions thereto provided that following such alterations to the sequence, the molecule is still capable of eliciting a specific protective immune response against the naturally occurring Helicobacter antigen. A similar immunogenic requirement is necessary for any fragments or derivatives of the antigen whether made from the recombinant molecule or the naturally occurring molecule. Accordingly, reference herein to a Helicobacter antigen is considered reference to the naturally occurring molecule, its recombinant form and any mutants, derivatives, fragments, homologues or analogues thereof provided that such molecules elicit a specific protective immune response against the naturally occurring Helicobacter antigen. Also included are fusion molecules between two or more Helicobacter antigens or with other molecules including fusion molecules with other molecules such as glutathione-S-transferase (GST) or xcex2-galactosidase.
The present invention also extends to an isolated nucleic acid molecule encoding a Helicobacter catalase antigen and preferably having a nucleotide sequence as set forth in SEQ ID NO. 1 or 3, or being substantially similar to all or a part thereof. The term xe2x80x9csubstantially similarxe2x80x9d means having at least 40-50%, more preferably at least 60-70%, and most preferably at least 80% identity. A xe2x80x9cpartxe2x80x9d in this context means a contiguous series of at least 15 nucleotides, and more preferably at least 25 nucleotides.
According to this embodiment, there is provided a nucleic acid molecule comprising a sequence of nucleotides which encodes a Helicobacter catalase antigen and hybridises under low stringency conditions to all or part of a nucleic acid sequence set forth in SEQ ID NO. 1 or 3, or to a complementary form thereof.
In another aspect, this invention provides a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO. 1 or 3, or a part thereof.
The nucleic acid molecule may be RNA or DNA, single stranded or double stranded, in linear or covalently closed circular form. For the purposes of defining the level of stringency, reference can conveniently be made to moeity (1982) at pp 387-389 which is herein incorporated by reference where the washing step at paragraph 11 is considered high stringency. A low stringency is defined herein as being in 0.1-0.5 w/v SDS at 37-45xc2x0 C. for 2-3 hours. Depending on the source and concentration of nucleic acid involved in the hybridisation, alternative conditions of stringency may be employed such as medium stringent conditions which are considered herein to be 0.25-0.5% w/v SDS at xe2x89xa745xc2x0 C. for 2-3 hours or high stringent conditions as disclosed by moeity (1982).
It will be appreciated that the sequence of nucleotides of this aspect of the invention may be obtained from natural, synthetic or semi-synthetic sources; furthermore, this nucleotide sequence may be a naturally-occurring sequence, or it may be related by mutation, including single or multiple base substitutions, deletions, insertions and inversions, to such a naturally-occurring sequence, provided always that the nucleic acid molecule comprising such a sequence is capable of being expressed as a Helicobacter antigen as broadly described above.
The nucleotide sequence may have expression control sequences positioned adjacent to it, such control sequences usually being derived from a heterologous source.
This invention also provides a recombinant DNA molecule comprising an expression control sequence having promoter sequences and initiator sequences and a nucleotide sequence which codes for a Helicobacter catalase antigen, the nucleotide sequence being located 3xe2x80x2 to the promoter and initiator sequences. In yet another aspect, the invention provides a recombinant DNA cloning vehicle capable of expressing a Helicobacter catalase antigen comprising an expression control sequence having promoter sequences and initiator sequences, and a nucleotide sequence which codes for a Helicobacter catalase antigen, the nucleotide sequence being located 3xe2x80x2 to the promoter and initiator sequences. In a further aspect, there is provided a host cell containing a recombinant DNA cloning vehicle and/or a recombinant DNA molecule as described above.
Suitable expression control sequences and host cell/cloning vehicle combinations are well known in the art, and are described by way of example, in moeity (1982).
In yet further aspects, there is provided fused polypeptides comprising a Helicobacter catalase antigen of this invention and an additional polypeptide, for example a polypeptide coded for by the DNA of a cloning vehicle, fused thereto. Such a fused polypeptide can be produced by a host cell transformed or infected with a recombinant DNA cloning vehicle as described above, and it can be subsequently isolated from the host cell to provide the fused polypeptide substantially free of other host cell proteins.
The present invention also extends to synthetic polypeptides displaying the antigenicity of a Helicobacter catalase antigen of this invention. As used herein, the term xe2x80x9csyntheticxe2x80x9d means that the polypeptides have been produced by chemical or biological means, such as by means of chemical synthesis or by recombinant DNA techniques leading to biological synthesis. Such polypeptides can, of course, be obtained by cleavage of a fused polypeptide as described above and separation of the desired polypeptide from the additional polypeptide coded for by the DNA of the cloning vehicle by methods well known in the art. Alternatively, once the amino acid sequence of the desired polypeptide has been established, for example, by determination of the nucleotide sequence coding for the desired polypeptide, the polypeptide may be produced synthetically, for example by the well-known Merrifield solid-phase synthesis procedure.
Once recombinant DNA cloning vehicles and/or host cells expressing a Helicobacter catalase antigen of this invention have been identified, the expressed polypeptides synthesised by the host cells, for example, as a fusion protein, can be isolated substantially free of contaminating host cell components by techniques well known to those skilled in the art.
Isolated polypeptides comprising, or containing in part, amino acid sequences corresponding to a Helicobacter catalase antigen may be used to raise polyclonal antisera by immunising rabbits, mice or other animals using well established procedures. Alternatively, such polypeptides may be used in the preparation of monoclonal antibodies by techniques well known in the art.
In addition, the polypeptides in accordance with this invention including fused polypeptides may be used as an active immunogen in the preparation of single or multivalent vaccines by methods well known in the art of vaccine manufacture for use in the treatment or prevention of Helicobacter infection in a mammalian host.
Alternatively, the polypeptides in accordance with the present invention including fused polypeptides may be used as antigen in a diagnostic immunoassay for detection of antibodies to Helicobacter in a sample, for example, a serum sample from a human or other mammalian patient. Such immunoassays are well known in the art, and include assays such as radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA).
The present invention also extends to delivery to the host using a vector expressing the catalase of Helicobacter bacteria, or an immunogenic fragment thereof. Accordingly, in a further aspect this invention provides a preparation for use in the treatment or prevention of Helicobacter infection in a mammalian host, which comprises a vector expressing the catalase of Helicobacter bacteria or an immunogenic fragment thereof.
In this aspect, the invention extends to a method for the treatment or prevention of Helicobacter infection in a mammalian host, which comprises administration to said host of a vector expressing the catalase of Helicobacter bacteria or an immunogenic fragment thereof.
Further, the invention extends to the use of a vector expressing the catalase of Helicobacter bacteria or an immunogenic fragment thereof, for the treatment or prevention of Helicobacter infection in a mammalian host.
Throughout this specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, is to be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.